PROJECT SUMMARY Arrestin proteins are master regulators of G protein coupled receptor (GPCR) signaling, and act in two ways. First, arrestins terminate the coupling of G proteins to cognate receptor by physically blocking G protein coupling. Second, arrestins can support G protein independent signaling. Here, there are many mysteries in the field ? arrestin can potentially activate one of over 150 signaling proteins, how does it select the correct one? The best studied arrestin-mediated signaling cascades involving mitogen activated protein (MAP) kinases. Recently we determined the structure of activated arrestin-3 in a conformation that is biased toward activating the MAP kinase Jun N-terminal Kinase-3 (JNK3). This JNK3-biased arrestin-3 structure showed two types of conformational change: (1) the expected inter-domain twist, and (2) localized, previously unrecognized conformational changes in the effector binding regions of activated arrestin. Combining structural analysis with functional measurements, we leverage these results to propose how arrestin biases toward one particular pathway. We propose that these different twists and localized conformational changes in arrestin work together to form specific binding sites. The magnitude of these conformational changes would depend upon the identity and phosphorylation pattern of the receptor. Here, we test an extension of the ?bar code hypothesis?, which would suggest that the phosphorylation pattern of receptor can push the arrestin structure toward slightly different conformations. In Aim 1, we develop synthetic phosphopeptides that contain a site-specific photoactivatable crosslinker and different receptor phosphorylation patterns. We will crosslink these peptides to arrestin-3, monitor activation via DEER spectroscopy, and assess how the phosphorylation pattern affects binding to MAP kinases in vitro. We will further crystallize these irreversibly activated arrestins to identify how the conformations vary with the changes in phosphorylation pattern. In Aim2, we propose site-specific mutagenesis and chimeragenesis to systematically assess the contribution of two key points of contact between arrestin and receptor in biased signaling: the phosphate sensor and the activation sensor. These studies will use receptor coupling assays performed in cells.